Monoclonal antibody of odorobacter specific protein and application thereof

Abstract

The invention discloses a monoclonal antibody of odorobacter specific protein and application of the monoclonal antibody, and belongs to the technical field of biology. TIR and Porin protein on the surface of odorobacter are used as immunogens, a monoclonal antibody with high specificity and high antigen affinity is prepared through a hybridoma technology, and after the monoclonal antibody is coupled with magnetic beads, the obtained magnetic bead antibody conjugate can efficiently enrich and / or separate odorobacter in a to-be-treated sample, so that the detection sensitivity of the odorobacter is improved, and the detection sensitivity is improved. The advantages of convenience and low cost are realized; in addition, the separated odorobacter can be used for culture, qualitative and quantitative research on metabolites of the odorobacter, the relationship of interaction with other microorganisms and the like, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and particularly relates to a monoclonal antibody of Odoribacter specific protein and application thereof. BACKGROUND

[0002] Odoribacter is a kind of gram-negative anaerobic bacillus, belonging to Bacteroidetes. Its shape is mostly straight rod or slightly curved rod, and it has no spore. Odoribacter is a strict anaerobe, and its optimum growth temperature is 37℃, and its pH is neutral. Odoribacter can ferment various carbohydrates (such as glucose and lactose), produce acid and release sulfur compounds (such as hydrogen sulfide) and volatile fatty acids, and is named because the metabolic products have a special odor. Odoribacter widely exists in the intestines, oral cavity and natural environment (such as sewage and humus) of humans and animals, is a normal member of intestinal flora, participates in polysaccharide degradation and energy metabolism, and maintains the balance of intestinal microecology. In environmental management, Odoribacter can help sewage purification by degrading complex organic matter; in intestinal health research, the change in its abundance is related to inflammatory bowel disease, obesity and the like, and can be used as a biomarker of dysbiosis; and some strains can also be used for developing microbial preparations to assist in regulating intestinal function.

[0003] Since Odoribacter plays an important role in intestinal flora, its content in the intestine has a certain relationship with the health of the organism. At present, Odoribacter in biological fecal samples is often enriched, separated and detected to determine its influence on the health of the organism. Among them, the immunomagnetic bead method for separating Odoribacter in feces has been widely used. The main principle of the immunomagnetic bead method is to use magnetic beads coated with antibodies that specifically bind to the surface proteins of Odoribacter to selectively capture Odoribacter, and then complete the mechanical movement by magnetic force technology, thereby separating Odoribacter.

[0004] However, the existing antibodies that specifically bind to the surface proteins of Odoribacter have problems such as poor specificity and weak affinity. SUMMARY

[0005] The purpose of the present application is to provide a monoclonal antibody of Odoribacter specific protein and application thereof. In the present application, the surface TIR and Porin proteins of Odoribacter are used as immunogens to prepare a monoclonal antibody with strong specificity and high affinity to the antigen. After coupling the monoclonal antibody with magnetic beads, the obtained magnetic bead antibody conjugate can realize efficient enrichment and / or separation of Odoribacter.

[0006] In a first aspect, the present application provides a monoclonal antibody or antigen-binding fragment thereof of a specific protein of the genus Odoribacter, comprising a heavy chain variable region and a light chain variable region as shown in A1) or A2) below; A1) the heavy chain variable region comprises complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 having the amino acid sequences shown in SEQ ID NOs: 13-15, respectively, and the light chain variable region comprises complementarity determining regions CDR-L1, CDR-L2 and CDR-L3 having the amino acid sequences shown in SEQ ID NOs: 17-19, respectively; A2) the heavy chain variable region comprises complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 having the amino acid sequences shown in SEQ ID NOs: 21-23, respectively, and the light chain variable region comprises complementarity determining regions CDR-L1, CDR-L2 and CDR-L3 having the amino acid sequences shown in SEQ ID NOs: 25-27, respectively.

[0007] In the monoclonal antibody or antigen-binding fragment thereof of a specific protein of the genus Odoribacter provided by the present application, the antigen-binding fragment comprises at least one of Fab, Fab', F(ab')2, antibody variable region (Fv), disulfide-stabilized Fv (dsFv), single-chain antibody (ScFv), and single-domain antibody (sdAb).

[0008] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof in A1) targets and binds to a TIR protein on the surface of the genus Odoribacter, and the monoclonal antibody or antigen-binding fragment thereof in A2) targets and binds to a Porin protein on the surface of the genus Odoribacter.

[0009] In some embodiments, the amino acid sequence of the TIR protein is shown in SEQ ID NO: 1, and the nucleotide sequence of a codon-optimized gene encoding the TIR protein is shown in SEQ ID NO: 2; the amino acid sequence of the Porin protein is shown in SEQ ID NO: 5, and the nucleotide sequence of a codon-optimized gene encoding the Porin protein is shown in SEQ ID NO: 6.

[0010] The above-mentioned TIR and Porin proteins provided by the present application can be natural, recombinant or synthetic active polypeptides, which can be natural purified products, chemically synthesized products, or products produced using recombinant technology from prokaryotic hosts (e.g., Escherichia coli) or eukaryotic hosts (e.g., yeast, higher plants).

[0011] In some embodiments, the above-mentioned TIR and Porin proteins are obtained by introducing a recombinant vector containing a gene encoding the same into an expression host (e.g., Escherichia coli BL21 (DE3)) to obtain a recombinant genetically engineered strain, then culturing the recombinant genetically engineered strain and inducing expression to obtain the TIR and Porin proteins.

[0012] In the present application, the monoclonal antibody or antigen-binding fragment thereof of the Odoribacter-specific protein can target and bind to the surface TIR and Porin protein of Odoribacter, and has the advantages of high specificity and high affinity.

[0013] In some embodiments, in A1), the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 16, or an amino acid sequence having 80% or more sequence identity to the sequence shown in SEQ ID NO: 16; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 20, or an amino acid sequence having 80% or more sequence identity to the sequence shown in SEQ ID NO: 20; in A2), the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 24, or an amino acid sequence having 80% or more sequence identity to the sequence shown in SEQ ID NO: 24; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 28, or an amino acid sequence having 80% or more sequence identity to the sequence shown in SEQ ID NO: 28.

[0014] As used herein, the term "sequence identity" can be evaluated by eye or by computer software, such as the software program described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of identity between two or more sequences can be expressed as a percentage (%) which can be used to evaluate the identity between related sequences. A polynucleotide sequence or amino acid sequence has a certain percentage (e.g. 90%, 95%, 98% or 99%) of "sequence identity" to another sequence if that percentage of bases or amino acids are the same in the two sequences when aligned.

[0015] In a second aspect, the present application provides a biological material selected from any one of the following: B1) a nucleic acid molecule encoding any one of the above monoclonal antibodies or antigen-binding fragments thereof; B2) a recombinant vector containing the nucleic acid molecule of B1); B3) a recombinant cell containing the nucleic acid molecule of B1) or the recombinant vector of B2).

[0016] The above-mentioned nucleic acid molecule provided by the present application can be DNA, such as cDNA, genomic DNA or recombinant DNA; or RNA, such as mRNA or hnRNA, etc.; and the nucleic acid molecule can generally be obtained by PCR amplification or artificial synthesis.

[0017] The recombinant vector provided by this invention includes a cloning vector and an expression vector. The cloning vector is used to replicate the relevant sequence, and the expression vector is used to express the relevant gene. The vector used to construct the expression vector can be a pET28a(+) vector.

[0018] In a third aspect, the present invention provides a magnetic bead antibody conjugate comprising a magnetic bead and any of the above-described monoclonal antibodies or antigen-binding fragments thereof conjugated to the magnetic bead.

[0019] The magnetic bead antibody conjugate provided by this invention can achieve efficient enrichment and / or separation of *Ostomyces* spp.

[0020] In a fourth aspect, the present invention provides a method for preparing a magnetic bead antibody conjugate, comprising the following steps: diluting any of the above-mentioned monoclonal antibodies or their antigen-binding fragments to obtain a diluent of the monoclonal antibody or its antigen-binding fragment; performing carboxyl activation treatment on magnetic beads to obtain activated carboxyl magnetic beads; and conjugating the diluent of the monoclonal antibody or its antigen-binding fragments with the activated carboxyl magnetic beads to obtain a magnetic bead antibody conjugate.

[0021] In some embodiments, in the step of diluting any of the above monoclonal antibodies or their antigen-binding fragments, a buffer solution is used for dilution, and the buffer solution is preferably MES buffer with a concentration of 10-20 mM, preferably 15 mM.

[0022] In some embodiments, in the step of carboxyl activation treatment of the magnetic beads, EDC solution and NHS solution are used for activation, wherein the concentration of EDC solution is 15-25 mg / mL, preferably 20 mg / mL; the concentration of NHS solution is 20-30 mg / mL, preferably 24 mg / mL; the activation treatment includes: activation at a temperature of 20-30°C (preferably 25°C) for 25-35 min, preferably 30 min.

[0023] In some embodiments, in the step of coupling the monoclonal antibody or its antigen-binding fragment dilution with the activated carboxyl magnetic beads, the molar ratio of the monoclonal antibody or its antigen-binding fragment dilution to the activated carboxyl magnetic beads is 1:(5-10), and the diameter of the activated carboxyl magnetic beads is 50-1000 nm.

[0024] In some implementations, the diameter of the activated carboxyl magnetic beads is 200 nm.

[0025] In this invention, by controlling the parameters in the preparation process of magnetic bead antibody conjugates within a specific range, the performance of the magnetic bead antibody conjugates can be improved, facilitating more efficient enrichment and / or separation of Osmotherium spp.

[0026] In a fifth aspect, the present application provides use of the monoclonal antibody or antigen binding fragment thereof of any one of the above, the biomaterial of the above, the magnetic bead antibody conjugate of the above, or the magnetic bead antibody conjugate prepared by the preparation method of any one of the above in enrichment and / or separation of the genus Odoribacter.

[0027] In a sixth aspect, the present application provides a method for enriching and / or separating the genus Odoribacter using the magnetic bead antibody conjugate of the above or the magnetic bead antibody conjugate prepared by the preparation method of any one of the above, comprising the following steps: providing a sample to be treated; adding the magnetic bead antibody conjugate to the sample to be treated, and after incubation, separating to obtain magnetic beads combined with the genus Odoribacter; suspending the magnetic beads combined with the genus Odoribacter in a solution, adding a protease and incubating, and after separation, obtaining a suspension of the genus Odoribacter.

[0028] In some embodiments, the sample to be treated comprises a fecal sample.

[0029] It can be understood that the sample to be treated can be routinely selected according to actual needs, as long as it contains the genus Odoribacter to be enriched and / or separated. For example, in the present application, the sample to be treated preferably comprises a fecal sample.

[0030] In some embodiments, in the step of adding the magnetic bead antibody conjugate to the sample to be treated and after incubation, the incubation specifically comprises: incubating at a temperature of 30-45°C (preferably 37°C) for 1-3h, preferably 2h.

[0031] In some embodiments, in the step of suspending the magnetic beads combined with the genus Odoribacter in a solution, adding a protease and incubating, the amount of the protease added is 0.05%, and the protease comprises papain; the incubation specifically comprises: incubating at a temperature of 30-45°C (preferably 37°C) for 2-4h, preferably 3h.

[0032] It can be understood that the type and amount of the protease can be routinely adjusted according to actual needs, as long as it has high Fc and Fab fragment cleavage activity. For example, in the present application, the amount of the protease added is preferably 0.05%, and the protease preferably comprises papain.

[0033] The beneficial effects of the present application are: different from the prior art, the present application takes odor bacillus surface TIR and Porin protein as immunogen, and specific monoclonal antibody with high antigen affinity is prepared by hybridoma technology, after coupling the monoclonal antibody and magnetic beads, the magnetic bead antibody conjugate obtained can realize efficient enrichment and / or separation of odor bacillus in the sample to be treated, and has the advantages of convenience and low cost; in addition, the separated odor bacillus can be used for culture, qualitative and quantitative research of its metabolites and the relationship with other microorganisms, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 SDS-PAGE detection results of the TIR and Porin proteins expressed and purified in the embodiment 1 of the present application; Figure 2 The titer detection results of the monoclonal antibody 3C7 in the embodiment 2 of the present application; Figure 3 The titer detection results of the monoclonal antibody 5E3 in the embodiment 2 of the present application; Figure 4 The SDS-PAGE detection results of the purified monoclonal antibodies 3C7 and 5E3 in the embodiment 2 of the present application; Figure 5 The WB detection results of the monoclonal antibody 3C7 specifically recognizing TIR protein in the embodiment 2 of the present application; Figure 6 The WB detection results of the monoclonal antibody 5E3 specifically recognizing Porin protein in the embodiment 2 of the present application. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be described in detail below, obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0036] The experimental methods not specified in the embodiments are usually carried out according to the conventional experimental methods in the field of molecular biology, including but not limited to the experimental methods described in Molecular Cloning: A Laboratory Manual by M.R. Green, Molecular Biology by Robert·F·Weaver, or the experimental methods suggested by the manufacturers of reagent kits and instrument equipment. The reagents and biological materials used in the embodiments can be obtained from commercial channels if not otherwise specified.

[0037] Preparation of TIR and Porin proteins on the surface of Odoribacter 1.1 Construction of TIR and Porin recombinant expression vectors Bioinformatics analysis of TIR protein (NCBI, protein_id: WP_181574395.1) showed that the intracellular region of TIR protein was 1-186 amino acids (aa), the transmembrane region was 187-204 amino acids (aa), and the extracellular region was 205-1066 amino acids (aa). The extracellular region was expressed in vitro. The amino acid sequence of TIR protein is shown in SEQ ID NO: 1, and the nucleotide sequence of the TIR gene synthesized after codon optimization is shown in SEQ ID NO: 2.

[0038] Similarly, bioinformatics analysis of Porin protein (NCBI, protein_id: WP_046451289.1) showed that Porin protein was composed of a signal peptide (1-17 amino acids (aa)) and an extracellular region. The extracellular region was selected as the 18-215 amino acid (aa) sequence for in vitro prokaryotic expression. The amino acid sequence of Porin protein is shown in SEQ ID NO: 5, and the nucleotide sequence of the Porin gene synthesized after codon optimization is shown in SEQ ID NO: 6.

[0039] Primers for amplifying TIR and Porin genes were designed and synthesized, and the specific sequences are as follows: TIR-F: 5'-TTTTGAATTCGATTATTACCGTACCAAGAA-3' (SEQ ID NO: 3); TIR-R: 5'-TTTTAAGCTTCTGGATAATTTCATTCACAT-3' (SEQ ID NO: 4); Porin-F: 5'-TTTTGAATTCTCTGGTCAGCTGGTTCGCCG-3' (SEQ ID NO: 7); Porin-R: 5'-TTTTAAGCTTGTCGCAACCGCAGTCACCAA-3' (SEQ ID NO: 8); The above primers contain EcoRI and HindIII restriction sites.

[0040] The gene amplification operation was carried out according to the following steps: a) Using the primers designed and synthesized above, TIR and Porin genes were amplified by PCR technology, respectively; b) After the completion of the PCR reaction, the PCR product was purified by gel recovery method to remove the excess impurities; c) The PCR product and the vector pET28a(+) were double-digested by EcoRI and HindIII respectively to generate sticky ends which could be ligated complementarily; d) The digested products were purified to obtain the target fragment and the linearized vector.

[0041] The following system was used: TIR or Porin fragment: 1 μL; vector fragment: 3 μL; T4 DNA ligase: 1 μL; 10 x buffer: 1 μL; water: 4 μL; total volume: 10 μL; after mixing the components well, the mixture was incubated at 16 °C overnight to allow the TIR or Porin to be fully ligated with the vector fragment.

[0042] 5 μL of the ligation product was added to 100 μL of competent cells, mixed gently, and then incubated in ice bath for 30 minutes to allow the ligation product to fully contact the competent cells. Subsequently, the mixture was heat-shocked at 42 °C for 45 seconds to facilitate the entry of the DNA fragment into the competent cells, and then quickly subjected to ice bath for 3 minutes. 500 μL of LB medium was added, and the transformed cells were incubated at 37 °C for 1 hour to recover the growth ability. Finally, the culture was spread on an LB agar plate containing 50 μg / mL kanamycin, and incubated at 37 °C for 12-16 hours to obtain single colonies of successful transformation.

[0043] Three single colonies were randomly selected from the LB agar plate, inoculated into 2 mL of LB liquid medium containing 50 μg / mL kanamycin, and incubated at 37 °C, 220 rpm overnight. After the incubation, the plasmid in each bacterial solution was extracted, double-digested by EcoRI and HindIII, and verified by sequencing. According to the results of digestion and sequencing, the correctly constructed prokaryotic expression vectors pET28a(+)-TIR and pET28a(+)-Porin were selected for subsequent experimental research.

[0044] 1.2 Expression and purification of TIR and Porin proteins The expression and purification of TIR and Porin proteins were carried out according to the following steps: Transformation culture: the recombinant plasmids pET28a(+)-TIR and pET28a(+)-Porin confirmed by sequencing in step 1.1 were transformed into E. coli BL21 (DE3) competent cells, respectively. The transformed cells were inoculated into LB solid medium containing 50 μg / mL kanamycin, and incubated at 37 °C for 12-16 hours to obtain resistant colonies.

[0045] Seed culture: Single colony was selected and inoculated into 2 mL LB liquid medium containing 50 pg / mL kanamycin, and cultured at 37 °C, 220 rpm overnight. The next day, the seed culture was inoculated into 200 mL LB medium containing the same concentration of kanamycin at a ratio of 1:200, and cultured at 37 °C, 220 rpm until OD 600 reached 0.6-0.8.

[0046] Protein induction expression: After culturing to the appropriate optical density, IPTG was added to the culture medium to achieve a final concentration of 1 mM. Subsequently, the culture temperature was maintained at 37 °C, and the expression was induced at 220 rpm for 16 hours. After the induction was completed, the culture was placed in a 4 °C environment, centrifuged at a speed of 12,000 x g for 1 minute, the supernatant was discarded, and the bacterial pellet was collected for use.

[0047] Cell disruption: After resuspending the collected bacterial pellet with PBS solution, it was treated with ultrasonic wave disruption method (5 seconds of work / interval each, total duration of 10 minutes) until the suspension was clear. Subsequently, it was centrifuged at 12,000 x g for 10 minutes at 4 °C, the supernatant was collected and filtered through a 0.22 pm filter membrane.

[0048] Nickel column chromatography: Column loading: After the Ni-NTA agarose gel packing material (GE Healthcare) was fully suspended, it was loaded into a chromatography column, and the column was washed with deionized water at a flow rate of 1-2 mL / min, with a volume of 5-10 column volumes.

[0049] Equilibration: The column was equilibrated with equilibration buffer (20 mM Na2HPO4, 0.5 M NaCl, pH 7.4) until the conductivity of the effluent was stable.

[0050] Sample loading: The filtered sample was loaded onto the nickel column at a flow rate of 0.5 mL / min to ensure that the target protein with His tag was fully bound.

[0051] Washing: The column was washed with equilibration buffer containing 20 mM imidazole to remove non-specifically bound impurities, and the absorbance at 280 nm was monitored until the baseline was stable.

[0052] Elution: The target protein was eluted stepwise with equilibration buffer containing 250 mM imidazole, and the elution peak was collected.

[0053] Post-treatment: The collected eluate was subjected to PBS dialysis treatment (4 °C overnight) to remove imidazole, and then the protein was concentrated by ultrafiltration (Amicon Ultra-15, 10 kDa cutoff). Finally, the purity of the purified protein was analyzed by SDS-PAGE.

[0054] SDS-PAGE detection results are shown in Figure 1

[0055] As can be seen from Figure 1 , the TIR and Porin proteins are consistent with the expected molecular weight size. Among them, the purity of TIR protein reaches 89%, and the concentration is 2.1 mg / mL; the purity of Porin protein reaches 91%, and the concentration is 1.5 mg / mL.

[0056] Example 2 Preparation of monoclonal antibody targeting TIR and Porin proteins on the surface of Brevundimonas 2.1 Mouse immunization The TIR and Porin proteins prepared in Example 1 were mixed with Freund's complete adjuvant, respectively, and then emulsified in a homogenizer. The first immunization was intraperitoneal and subcutaneous injection, and the immunization dose was 100 μg of protein. The second to fourth immunizations were all subcutaneous injection, and the immunization dose was 50 μg of protein. The immunization was performed every two weeks, and after four times of immunization, the tail vein blood of the mouse was taken, and the serum antibody titer was detected. The mouse with the highest titer was selected for the preparation of subsequent hybridoma cells.

[0057] 2.2 Preparation of hybridoma cells 1) The mouse with the highest antibody titer after immunization was taken, and the spleen cell suspension was prepared and washed with PBS. Then the SP2 / 0 cells were mixed, the spleen cells: SP2 / 0 = 7:1, centrifuged at 1500 rpm for 5 min, and then the mixed cells were dripped and the cell mass was gently knocked. 1 mL of PEG 1450 preheated at 37°C was added, and after addition, it was reacted in a 37°C water bath for 1 min, and 40 mL of RPMI-1640 termination liquid was slowly added along the tube wall.

[0058] 2) After the termination of the fused cells, centrifugation was performed at 800 rpm for 5 min, and the residual liquid was aspirated. The cells were resuspended in 100 mL of HAT medium containing 20% FBS, and then plated into a 96-well cell culture plate containing feeder cells, and cultured in a 37°C, 5% CO2 carbon dioxide incubator.

[0059] 3) After 8-10 days of fusion, the cell state was observed, the supernatant titer of the cells was determined by indirect ELISA method, and the positive cell strain with high titer and good specificity was selected for subcloning.

[0060] 4) The cells screened by the HT medium were diluted to 1 cell / well by limiting dilution method, and then plated into a 96-well cell culture plate. When the monoclonal cells grew to a medium size, the density was about 10 4 ​The titer can be detected above one cell, and then the positive cells are taken again, and the subcloning screening is repeated once. When the supernatant of the cells in all micropores is positive, the hybridoma cell strain is obtained after three subcloning, and the cell strain targeting the binding of TIR protein is named hybridoma cell strain 3C7, and the cell strain targeting the binding of Porin protein is named hybridoma cell strain 5E3. The secreted antibodies are named monoclonal antibodies 3C7 and 5E3, respectively. The titers of the above-mentioned monoclonal antibodies are all above 1:320000, and the detection results are shown in Table 1 and Figures 2-3 as shown.

[0061] Table 1 Detection results of monoclonal antibody 3C7 and 5E3 titers

[0062] 2.3 Purification of monoclonal antibodies The hybridoma cell strains 3C7 and 5E3 obtained in step 2.2 are resuscitated in the presence of serum, and during several consecutive cell passages, the proportion of serum is gradually reduced (such as 20% serum to 15% serum to 10% serum to serum-free in turn), and the culture medium is gradually increased, so that the hybridoma cells gradually adapt to the serum-free culture environment.

[0063] In the preparation of monoclonal antibodies, the hybridoma cell strains 3C7 and 5E3 obtained in step 2.2 are resuscitated in serum-free medium, and when the cells are in good condition, the amount of culture medium and the container for culturing cells are increased. When the cells die in large quantities, the liquid is collected and centrifuged to obtain the supernatant, filtered and collected, and the sample to be purified is loaded onto a ProteinA-agarose affinity chromatography column at a flow rate of 0.5 mL / min. The antibody is combined with ProteinA, and finally eluted with an eluent to obtain the purified antibody. The purity of the antibody is identified by SDS-PAGE, and the results are shown in Figure 4 as shown.

[0064] As can be seen from Figure 4 , the sizes of the monoclonal antibodies 3C7 and 5E3 are consistent with the expected, and have high purity.

[0065] 2.4 Specificity analysis of monoclonal antibodies The specificity of the monoclonal antibodies is verified by Western Blot technology. Specifically, the following steps are included: 1) Polyacrylamide gel electrophoresis (SDS-PAGE) Gel preparation: A vertical electrophoresis system of 10% separation gel and 5% concentration gel is used.

[0066] Loading parameters: Pre-stained protein Marker (5 μL) and sample to be tested (20 μL) are loaded, and empty load BL21(DE3) lysate is used as negative control (NC).

[0067] Electrophoresis procedure: Start at a constant voltage of 80V, and increase the voltage to 120V after the bromophenol blue indicator enters the separating gel. The entire process takes about 2 hours.

[0068] 2) Transfer operation Membrane treatment: Cut a PVDF membrane to match the size of the gel and activate it with methanol for 5 minutes.

[0069] Sandwich assembly: Assemble in the following order: anode plate → filter paper → gel → PVDF membrane → filter paper → cathode plate, ensuring no air bubbles.

[0070] Electrotransfer conditions: Wet transfer at a constant current of 300mA for 1 hour, followed by washing with TBST buffer 3 times for 5 minutes each time.

[0071] 3) Immunoblotting detection Blocking: Block overnight at 4°C with 5% BSA-TBST solution.

[0072] Primary antibody incubation: Incubate 1A5 and 4D6 monoclonal antibodies diluted with 1% BSA-TBST (1:1000) at 37°C for 1 hour.

[0073] Secondary antibody incubation: Goat anti-mouse HRP-labeled antibody (1:20000 dilution), reacted at 37°C for 1 hour.

[0074] Washing procedure: 3 washes with TBST buffer, 10 minutes each time; 3 washes with PBST buffer, 10 minutes each time.

[0075] 4) Color imaging ECL chemiluminescence method for color development, and electrochemiluminescence imaging system for recording experimental results.

[0076] The results are as follows Figure 5 and 6 As shown.

[0077] from Figure 5 and 6 As can be seen, monoclonal antibody 3C7 specifically recognizes TIR protein, and monoclonal antibody 5E3 specifically recognizes Porin protein, both showing a single immune response band, confirming the high specificity of monoclonal antibodies.

[0078] 2.5 Sequencing of monoclonal antibodies The hybridoma cell strains 3C7 and 5E3 obtained in step 2.2 were respectively cultured, lysed, and total RNA was extracted. The mRNA was reversely transcribed to synthesize cDNA by using random hexamer primers (5'-P-d(NNNNNN)-3'; wherein, N=G, A, T or C), and then two rounds of nested PCR were performed: the first-strand cDNA was used as a template for amplification, the forward primer was complementary to the sequence of the corresponding heavy chain and light chain leader sequence, and the reverse primer was a sequence in the constant region of the heavy chain and light chain. The specific primer sequences are as follows: Heavy chain forward primer: 5'-CGGCCCAGCCGGCC-3' (SEQ ID NO: 9); Heavy chain reverse primer: 5'-TGAACCGCCTCCACC-3' (SEQ ID NO: 10); Light chain forward primer: 5'-GGTTCCACTGGT-3' (SEQ ID NO: 11); Light chain reverse primer: 5'-GTGCAGCATCAGC-3' (SEQ ID NO: 12).

[0079] The PCR amplification program was as follows: denaturation at 94℃ for 2 min; denaturation at 94℃ for 20 s, annealing at 58℃ for 20 s, and extension at 72℃ for 60 s, for 40 cycles of extension; and final extension at 72℃ for 5 min.

[0080] The second round of amplification produced a gene product with a restriction enzyme cutting site (EcoRI and XhoI), which was connected to the pTIG cloning vector, and then sequencing and analysis were performed to obtain the heavy chain and light chain variable region sequences of the monoclonal antibodies 3C7 and 5E3, as follows: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 3C7 is as follows: EVPLQQSGAELVRPGSSVKISCKASASTFSKSWTQWVKQRPGQGLEWIGCILSETEDTCQTRHRAGKATLTSDKSSSTAYMQLSSLTSEDSAVFFCGQKVSSSAALSSMEAWGQGTSVIVSS (SEQ ID NO: 16).

[0081] Among them, the complementarity determining regions of the heavy chain variable region of the monoclonal antibody 3C7 are as follows: CDR-H1: KSWTQ (SEQ ID NO: 13); CDR-H2: CILSETEDTCQTRHRAG (SEQ ID NO: 14); CDR-H3: KVSSSAALSSMEA (SEQ ID NO: 15).

[0082] The amino acid sequence of the monoclonal antibody 3C7 heavy chain variable region is as follows: DIGGTQTPLSLPVSLGDQASISCKTTWSTIPQSTSTISKWYLQKPDQSPKLLIYRRCTKMLGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCTCTSKIPMLFGSGTKLEIK (SEQ ID NO: 20).

[0083] In which, the complementarity determining regions of the monoclonal antibody 3C7 heavy chain variable region are as follows: CDR-H1: GFTFSSYAM (SEQ ID NO: 13); CDR-H2: VIWGNGN (SEQ ID NO: 14); CDR-H3: RNYMSYSDLDV (SEQ ID NO: 15).

[0084] The amino acid sequence of the monoclonal antibody 5E3 heavy chain variable region is as follows: DVQLQESGPGLVKPSQSLSLTCTGTGYSITTEYAGQEIRQFPGNKLEWMGSIVSVAACQSVPMRSTIRSILRSTSKNQFFLQLNSVATEDTATYFCARTTALSSENASSEMSSWGQGTSVTVSS (SEQ ID NO: 24).

[0085] In which, the complementarity determining regions of the monoclonal antibody 5E3 heavy chain variable region are as follows: CDR-H1: TEYAGQ (SEQ ID NO: 21); CDR-H2: SIVSVAACQSVPMRST (SEQ ID NO: 22); CDR-H3: TTALSSENASSEMSS (SEQ ID NO: 23).

[0086] The amino acid sequence of the monoclonal antibody 5E3 light chain variable region is as follows: DVQMNQSPKMMSTSIKDIVSITCKGASECSTGAGWKQSKPGQSPKLLIYSAASTKHGVPDRFTGSGSGTDFTLTISSVQAEDLALYYCCCKYSCVSTFGGGTKLEIK (SEQ ID NO: 28).

[0087] The complementarity determining regions of the monoclonal antibody 5E3 light chain variable region are as follows: CDR-L1: KGASECSTGAG (SEQ ID NO: 25); CDR-L2: SAASTKH (SEQ ID NO: 26); CDR-L3: CCKYSCVST (SEQ ID NO: 27).

[0088] Example 3 Preparation of magnetic bead antibody conjugate and enrichment and separation of Odoribacter 3.1 Preparation of magnetic bead antibody conjugate First, the magnetic bead antibody conjugate is prepared, specifically including the following steps: 3.1.1 Monoclonal antibody dilution The monoclonal antibodies 3C7 and 5E3 obtained in Example 2 are diluted with MES buffer (concentration 15 mM, pH 6.0) to an antibody concentration of 2 mg / mL, to obtain a monoclonal antibody 3C7 diluent and a monoclonal antibody 5E3 diluent, respectively.

[0089] 3.1.2 Activation of carboxyl magnetic beads 1) Take 1 mg of carboxyl magnetic beads, wash with MES buffer for 3 times, and resuspend with 0.1 mL of MES buffer to 10 mg / mL; 2) Weigh EDC and NHS to be dissolved in MES buffer, the concentration of EDC solution is 20 mg / mL, and the concentration of NHS solution is 24 mg / mL, 50 µL of EDC solution and 50 µL of NHS solution are added to the magnetic beads treated in step 1), and activated at 25°C for 30 min to obtain activated carboxyl magnetic beads.

[0090] 3.1.3 Covalent coupling of magnetic beads and antibodies Take the antibody diluent obtained in step 3.1 according to the antibody-magnetic bead molar ratio of 1:10 or 1:5, and mix with 1 mg of activated carboxyl magnetic beads (diameter of 50 nm, 200 nm and 1000 nm, respectively), react at 25°C for 16 h, magnetically separate, aspirate the supernatant and simultaneously detect the remaining antibody content in the supernatant, calculate the amount and concentration of magnetic bead conjugated antibodies, continue to wash the magnetic beads with normal saline for two to three times, resuspend with normal saline, and obtain magnetic bead antibody conjugates 3C7 ①, 3C7 ②, 3C7 ③, 3C7 ④, 3C7 ⑤, 3C7 ⑥, 5E3 ①, 5E3 ②, 5E3 ③, 5E3 ④, 5E3 ⑤, 5E3 ⑥, respectively. The conjugated magnetic bead conditions are shown in Table 2.

[0091] 3.2 Enrichment and isolation of odoribacter The magnetic bead antibody conjugate prepared in 3.1 was then used to enrich and isolate odoribacter, which specifically included the following steps: 1) Preparation of fecal bacteria suspension Take 10 g of fecal sample and add physiological saline at a ratio of 1:5 (i.e. 5 mL of physiological saline per 1 g of feces). Then mix the feces and physiological saline thoroughly. First, use a 10-mesh sieve to preliminarily filter the mixture to remove larger fecal residues. Then use 30-mesh and 60-mesh sieves in sequence to further filter the bacterial suspension.

[0092] Centrifuge the filtered bacterial suspension at 8000 g for 5 minutes. After centrifugation, discard the supernatant and add 40 mL of physiological saline to the precipitate, mix thoroughly to resuspend the bacteria in the physiological saline.

[0093] Repeat the above steps of centrifugation, discarding the supernatant, and resuspension three times to obtain a relatively clean fecal bacterial suspension.

[0094] 2) Incubation of magnetic bead antibody conjugate with fecal suspension Take 5 mL of the prepared fecal suspension and add 1 mg of magnetic bead antibody conjugate prepared in 3.1. Mix and incubate the mixture at 37°C for 2 hours to allow the magnetic bead antibody conjugate to fully bind to odoribacter. After incubation, use a magnetic stand to separate the magnetic beads, and remove the unbound microorganisms and supernatant.

[0095] 3) Separation of magnetic beads from odoribacter Resuspend the magnetic beads (labeled magnetic beads) bound with odoribacter in physiological saline. Use antibody labeling removal reagent, i.e. 0.05% papain (product number S10011, source leaf biological), to mix with the labeled magnetic beads and incubate at 37°C for 3 hours. Papain can cut the Fc and Fab fragments of mouse monoclonal antibodies, thereby separating the magnetic beads from odoribacter.

[0096] 4) Counting of odoribacter and determination of conditions Use the magnetic stand to collect the magnetic beads again, and the resulting supernatant is the odoribacter suspension. Dilute the suspension and add it to a blood cell counting plate for counting under a microscope. The yield of enriched odoribacter is shown in Table 2.

[0097] Table 2 Antibody conjugated magnetic bead conditions and yield results

[0098] As can be seen from Table 2, the magnetic bead antibody conjugate containing the monoclonal antibodies 3C7 and 5E3 in the application has a good enrichment efficiency on Odoribacter; and the magnetic bead antibody conjugate containing the monoclonal antibodies 3C7 and 5E3 at the same time has the best enrichment efficiency. In addition, the enrichment of Odoribacter has a good efficiency under the condition that the diameter of the magnetic bead is 200 nm and the molar ratio of the antibody to the magnetic bead is 1:5. The magnetic bead antibody conjugate of the application has the advantages of high separation and enrichment yield and strong specificity in the enrichment of Odoribacter.

[0099] Example 4 Identification of Odoribacter The isolated and collected Odoribacter in Example 3 was diluted by 1000 times and 10000 times, respectively, and then inoculated on the prepared solid culture medium (tryptone: 10.0 g, beef extract powder: 8.0 g, yeast extract: 4.0 g, glucose: 20.0 g, triammonium citrate: 2.0 g, dipotassium hydrogen phosphate: 2.0 g, sodium acetate: 5.0 g, MnSO4·4H2O: 0.05 g, MgSO4·7H2O: 0.2 g, Tween-80: 1.0 mL, cysteine hydrochloride: 0.5 g, agar powder: 15.0 g (for solid culture, not added for liquid culture), distilled water: 1000 mL, pH adjustment: adjusted to 6.2-6.4 with NaOH before sterilization, sterilized at 121℃ for 15 minutes) in an anaerobic incubator for more than 3 days, 10 single colonies were picked and cultured in vitro under anaerobic conditions, DNA was extracted, and 16S rRNA gene sequencing was performed with bacterial DNA as a template, and the sequencing results were subjected to BLAST comparison in the NCBI database. The 16S rRNA gene sequence of the bacteria has a homology of 100% with Odoribacter splanchnicus, and the 16S RNA gene sequences of the 10 single colonies in the sequencing results are the same, so it is determined that the isolated bacteria are Odoribacter bacteria.

[0100] In summary, the monoclonal antibody with high specificity and high affinity to the antigen is prepared by using the surface TIR and Porin protein of Odoribacter as the immunogen and by using the hybridoma technology. After coupling the monoclonal antibody with the magnetic bead, the magnetic bead antibody conjugate obtained can realize efficient enrichment and / or separation of Odoribacter in the sample to be treated.

[0101] The above examples only express the embodiments of the application, which are described in detail, but cannot be understood as the limitation of the patent scope of the application. It should be noted that, for those skilled in the art, without departing from the concept of the application, a number of modifications and improvements can be made, which all belong to the protection scope of the application. Therefore, the protection scope of the patent of the application should be subject to the appended claims.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof of a *Ostomyces* genus-specific protein, characterized in that, Includes the heavy chain variable region and the light chain variable region shown in A1) or A2) below; A1) The heavy chain variable region includes complementarity-determining regions CDR-H1, CDR-H2 and CDR-H3 as shown in SEQ ID NO: 13-15, respectively, and the light chain variable region includes complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NO: 17-19, respectively. A2) The heavy chain variable region includes complementarity-determining regions CDR-H1, CDR-H2 and CDR-H3 as shown in SEQ ID NO: 21-23, respectively, and the light chain variable region includes complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NO: 25-27, respectively.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, In A1), the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 16, or an amino acid sequence having more than 80% sequence identity with the sequence shown in SEQ ID NO: 16; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 20, or an amino acid sequence having more than 80% sequence identity with the sequence shown in SEQ ID NO:

20. In A2), the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 24, or an amino acid sequence having more than 80% sequence identity with the sequence shown in SEQ ID NO: 24; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 28, or an amino acid sequence having more than 80% sequence identity with the sequence shown in SEQ ID NO:

28.

3. A biomaterial, characterized in that, The biomaterial is selected from any one of the following: B1) A nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-2; B2) A recombinant vector containing the nucleic acid molecules described in B1); B3) Recombinant cells containing the nucleic acid molecule described in B1) or the recombinant vector described in B2).

4. A magnetic bead antibody conjugate, characterized in that, The invention includes magnetic beads and a monoclonal antibody or antigen-binding fragment thereof conjugated to any one of claims 1-2.

5. A method for preparing a magnetic bead antibody conjugate, characterized in that, Includes the following steps: The monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-2 is diluted to obtain a diluted solution of the monoclonal antibody or its antigen-binding fragment. Activated carboxyl magnetic beads were obtained by carboxyl activation treatment of magnetic beads. The monoclonal antibody or its antigen-binding fragment dilution is coupled with the activated carboxyl magnetic beads to obtain a magnetic bead antibody conjugate.

6. The preparation method according to claim 5, characterized in that, In the step of coupling the monoclonal antibody or its antigen-binding fragment dilution with the activated carboxyl magnetic beads, the molar ratio of the monoclonal antibody or its antigen-binding fragment dilution to the activated carboxyl magnetic beads is 1:(5-10), and the diameter of the activated carboxyl magnetic beads is 50-1000 nm.

7. The preparation method according to claim 6, characterized in that, The activated carboxyl magnetic beads have a diameter of 200 nm.

8. The use of the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-2, the biomaterial as described in claim 3, the magnetic bead antibody conjugate as described in claim 4, and the magnetic bead antibody conjugate prepared by the preparation method as described in any one of claims 5-7 in the enrichment and / or isolation of *Ostomyces* spp.

9. A method for enriching and / or separating *Ostomyces* spp. using the magnetic bead antibody conjugate according to claim 4 or the preparation method according to any one of claims 5-7, characterized in that, Includes the following steps: Provide the sample to be processed; The magnetic bead antibody conjugate was added to the sample to be processed, and after incubation, magnetic beads bound to Bacillus spp. were separated. The magnetic beads containing *Ostomyces* were suspended in a solution, incubated with protease, and then separated to obtain a suspension of *Ostomyces*.

10. The method according to claim 9, characterized in that, In the step of suspending the magnetic beads conjugated with *Ostomyces* in a solution and incubating them with a protease, the amount of protease added is 0.05%, and the protease includes papain.

Images